Electrically active combinatorial chemical (EACC) chip for biochemical analyte detection

ABSTRACT

Apparatus and methods are disclosed for electrically active combinatorial-chemical (EACC) chips for biochemical analyte detection. An apparatus includes a substrate that has an array of regions defining multiple cells, wherein each of the cells includes a reaction cavity that contains multiple functional binding groups. A method of detecting an analyte providing the reaction cavity between a source and a drain or a pair of electrodes, applying a voltage and monitoring a parameter indicative of an analyte characteristic. A process of fabricating an EACC include bonding an analyte to the multiple functional binding groups of each reaction cavity, and forming an analyte sensing structure including the substrate.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Embodiments of the invention relate generally to the field of biologicaland/or chemical sensing. More particularly, embodiments of the inventionrelate to electrically active combinatorial-chemical (EACC) chips forbiochemical analyte detection.

2. Background Information

Currently, biological and chemical analyte detections are basedprimarily on specific interaction between analytes and their bindingpartners. To perform high throughput assays, a large number of molecularprobes need to be immobilized on a surface to form a microarray. Suchmicroarrays are sometimes referred to as bio-chips (e.g., protein chipsor gene chips). Preparing a large number of specific polymeric probes(e.g., antibodies or nucleic acids) is, however, both time-consuming andcostly. Moreover, immobilizing the polymeric probes in discrete smallsurface areas is technically difficult and expensive. It is desired tohave a more efficient approach to preparing and immobilizing probes.

Traditional approaches to making biochips involve chemically preparingpolymeric probes and then subsequently spotting the chemically preparedpolymeric probes on the chips. However, the minimum feature sizeattainable with these probes is typically >100 um for a protein chip(array), or >1 um for a gene chip (array). It is desired to have smallerfeature sizes available in the future. While higher density bio-chipsare clearly desirable from the perspective of both cost to manufactureand clinical efficiency, fabricating higher density bio-chips based onsmaller polymeric probe feature sizes is both technically challengingand time-consuming. It is desired to have an approach that will permitthe fabrication of chips based on smaller probe feature sizes.

Referring to FIGS. 1A and 1B, current biochips for direct analytedetection (antibody chips, DNA chips, aptamer chips) are based oninteractions of analytes with their polymeric binding partners (probes),each of the latter of which presents unique intra molecular bindingsites. Referring to FIG. 1A, a binding partner (probe) 110 isimmobilized on a substrate 120. The binding partner 110 then binds withan analyte 130, thereby enabling the detection of the analyte 130. Thisbinding approach is based on the principle of using a single, unique andlarge molecule for specific binding of analytes. This approach is highlyspecific and accurate, and generally involves small dimension(s). On theother hand, this approach is very costly and time-consuming because ofthe need to obtain analyte-specific probes or binding partners, and isgenerally inflexible. Also, as only known probes are used to detectknown analytes; but not-yet-identified analytes are undetectable. It isdesired, therefore, to have an approach that can detect unknownanalytes.

Referring to FIG. 1B, two different types of analytes 140, 150 aredispersed across a substrate 160 by a buffer solvent flow. The analytes140, 150 are spatially segregated across a surface of the substrate 160,thereby enabling separation of two different analytes 140, 150. Theresulting spatial segregation permits detection of individual analytes.Separation in this instance is based on the principle of buffer solventflow. This approach is low cost, fast, and flexible, but is lessspecific and less accurate than is desired, and it involves largedimension(s). Another technique might involve molecular migration in agel (electrophoresis) based on size and molecular weight.

Protein binding to a surface may be affected by the chemical property ofthe surface. In this way, protein chips with different binding surfaceshave been produced. Chromagraphic and spectrographic binding surfacetechnologies have also been evolving, wherein bio-chip detections aretypically read by optical methods. When the chip feature (spot) sizebecomes <1 um, however, optical detection becomes impractical. It isdesired to have an approach that enables detection and reading withhigher density bio-chips.

Electronic sensors for biomolecule detection have also beendemonstrated. Although such electronic sensors have the potential toovercome the spatial limitations of optical detection, electronicsensors by themselves do not appear to obviate the underlying featuresize limitations of the polymeric probe-analyte paradigm.

Self aligned monolayers have been demonstrated. The formation ofpatterned co-planar monolayers (which can be termed ultra thin films)and the use of those patterns to selectively bind colloidal catalysts &plate electroless metals selectively at high resolution are underinvestigation. Further research into the formation of ultra thin filmsfor the selective adhesion of various types of biological cells isongoing.

Heretofore, the requirements of a more efficient approach to preparingand immobilizing probes, smaller probe feature sizes, the ability todetect unknown analytes and the detection and reading of higher densitybio-chips have not been fully met. It is therefore desired providetechniques that meet these goals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The drawings accompanying and forming part of this specification areincluded to depict certain aspects of embodiments of the invention. Aclearer conception of the embodiments of the invention, and of thecomponents and operation of systems provided with embodiments of theinvention, will become more readily apparent by referring to theexemplary, and therefore nonlimiting, embodiments illustrated in thedrawings, wherein identical reference numerals designate the sameelements. The embodiments of the invention may be better understood byreference to one or more of these drawings in combination with thedescription presented herein. It should be noted that the featuresillustrated in the drawings are not necessarily drawn to scale. Briefdescriptions are provided below, followed a detailed description of thepreferred embodiments in view of the illustrative drawings.

FIGS. 1A and 1B illustrate conventional techniques for binding andseparating analytes.

FIG. 1C illustrates the use of a plurality of different binding groupsto detect an analyte, representing an embodiment of the invention.

FIG. 2 illustrates top plan and partial cross section views of acombinatorial-chemical chip, representing an embodiment of theinvention.

FIGS. 3A-3C illustrate a combinatorial printing head, a side view offilled reaction cavities and a side view of mixed self assembledmonolayers, respectively, representing embodiments of the invention.

FIG. 4 illustrates four self assembled monolayer chemical structuresmapped across a two dimensional array, representing an embodiment of theinvention.

FIG. 5 illustrates a group of four multi-chemical gradient areas,representing an embodiment of the invention.

FIGS. 6A and 6B illustrate structural diagrams of a field effect sensorand a capacitance/impedance sensor, respectively, representingembodiments of the invention.

FIG. 7 illustrates a structural diagram of a sensor forstatic-electrical or capacitance/impedance measurements, representing anembodiment of the invention.

FIGS. 8A-8C illustrate static-electrical detection of an analyte,representing an embodiment of the invention.

FIG. 9A illustrates a schematic representation of a substrate of siliconor glass modified with a self aligned monolayer oftrichlorophenylsilane, representing an embodiment of the invention.

FIG. 9B illustrates a schematic representation of a first self alignedmonolayer (SAM) of trichlorophenylsilane exposed to about 50 mJ ofultraviolet light at ˜250 nm in clean room air. (˜10% of dose to clearall phenyl groups), representing an embodiment of the invention.

FIG. 9C illustrates a schematic representation of co-planar self alignedmonolayers after initial exposure and formation of a second self alignedmonolayer (SAM2), representing an embodiment of the invention.

FIG. 9D illustrates a schematic representation of a final composition ofSAM, SAM2, & SAM3 example after using 2 exposures of about 50 mJ(initial exposure) and 100 mJ (subsequent exposure) of ultraviolet lightat ˜250 nm, representing an embodiment of the invention.

FIGS. 10A and 10B illustrate DNA-based self-assembly examples,representing embodiments of the invention.

FIG. 11 illustrates a cross-linked polymer example, representing anembodiment of the invention.

FIGS. 12A and 12B illustrate co-polymerization and chain transferexamples, respectively, representing embodiments of the invention.

FIGS. 13A-13D illustrate thiol-PEG based examples, representingembodiments of the invention.

The embodiments of the invention and the various features andadvantageous details thereof are explained more fully with reference tothese non-limiting embodiments that are illustrated in the accompanyingdrawings and detailed in the following description. Descriptions of wellknown starting materials, processing techniques, components andequipment are omitted so as not to unnecessarily obscure the embodimentsof the invention in detail. It should be understood, however, that thedetailed description and the specific examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly and not by way of limitation. Various substitutions, modifications,additions and/or rearrangements within the spirit and/or scope of theunderlying inventive concept will become apparent to those skilled inthe art from this disclosure.

The descriptions herein of the invention and preferred and alternativeembodiments may be better understood in view of the followingdefinitions:

The term “non-polymer” refers to polyatomic organic molecules that donot have repeated units that are either identical or non-identical.

The term “protein chip” refers to a two or three dimensional device thatcontains immobilized protein species (2 or more proteins) that arearranged in regular patterns or irregular patterns.

The term “optical sensing structure” refers to a device that collectsphotons from other objects and converts them to electrical signals.

The term “reaction cavity” refers to a 3D space that can hold reactantsand allow chemical or biochemical reactions to proceed, typicallymeasured in nanometer or micrometer scales.

The term “feature size” refers to the dimension(s) of an individualfeature of a given array. For example, a protein array may have 100protein spots. Thus the protein spots are the features of the proteinarray. The dimension of a given spot is the feature size of the spot. Itcan be measured by area, diameter or lengths of sides.

The phrase “coupling via thiol-based reaction product” refers tocovalent bonding formation involving a hydrosulfide group (—SH). It canhappen between organic compounds or between a thiol-containing organiccompound and metals, such as gold and silver.

A substrate of an apparatus in accordance with a preferred embodimentincludes an array of regions defining multiple cells. Each of the cellsincludes a reaction cavity that contains multiple functional bindinggroups. The substrate includes a solid material that provides support aswell as a functional surface. The substrate can be made up of any ofseveral materials, and preferably inorganic materials such as siliconwafer, glass, metal (aluminum, e.g.), or organic material such asplastic (polycarbonate, e.g.). The surface of the substrate ispreferably coated with metal (gold) or a polymer (PEG) or both.Functional groups on the surface may include amine groups or carboxylgroups.

The multiple functional binding groups may be coupled to the substratevia hybridized DNA, a cross-linked polymer, a copolymer, a chaintransfer polymer and/or a thiol-based reaction product. The cellspreferably each include an analyte sensing structure such as anelectrical sensing circuit or an optical sensing structure.

The cells may each comprise a protein chip or gene chip having a featuresize preferably between 0.5 microns and 500 microns, and preferably lessthan approximately 100 microns. The cells may each comprise anelectrically-active, combinatorial-chemical (EACC) chip for biochemicalanalyte detection. The analyte detection may be probe-less. The groupsmay include non-polymeric components.

The array may include a first density gradient of a first group in afirst direction, and may further include a second density gradient of asecond group in a second direction. The second direction may beapproximately orthogonal to the first direction. Moreover, foursignificant directions may include, e.g., from an overhead viewpoint,left to right, right to left, up to down and down to up.

The substrate may comprise silicon having a surface modified withsilanes, wherein the silanes may comprise phenyl. The multiple groupsmay include a positively-charged group and a negatively charged groupand/or a polar group and a non-polar group.

A method of detecting an analyte uses a substrate including an array ofregions defining multiple cells. Each of the cells includes a reactioncavity containing multiple functional binding groups. A channel may bedefined between a source and a drain, although not necessarily, or aregion may be defined between a pair of electrodes. A voltage is appliedbetween the source and the drain or the pair of electrodes. A parameterindicative of an analyte characteristic is monitored when the voltage isapplied. Each of the cells may include an analyte bonded to aself-assembled monolayer to define a channel or region between a sourceand drain or pair of electrodes, respectively.

A method of making an analyte sensor uses a substrate including an arrayof regions defining a plurality of cells each including a reactioncavity. Multiple functional binding groups are coupled to each reactioncavity. An analyte sensing structure is formed including the substratewith the array of regions. An analyte is preferably bonded to themultiple functional binding groups of each reaction cavity.

The forming of the analyte sensing structure may include forming asource and a drain for each reaction cavity such that each reactioncavity may define, although not necessarily, a channel between thesource and the drain, and coupling a voltage source and monitoringsystem between the source and the drain, or it may include forming apair of electrodes for each reaction cavity, and coupling a voltagesource and monitoring system between the pair of electrodes. It may alsoinclude forming an optical sensing structure.

The method may include modifying a surface of the substrate withsilanes, and the silanes may comprise phenyl. Modifications methods mayinclude any of a variety of techniques such as adsorption or chargeinteraction.

A first gradient of a first group may be formed in a first direction ofthe array, and a second gradient of a second group may be formed in asecond direction of the array. The first and second directions may beorthogonal. Third and fourth directions would include those opposite tothe first and second directions.

An apparatus in accordance with an embodiment of the invention includesa substrate that includes an array of regions defining multiple cells,wherein each of the cells includes a reaction cavity that containsmultiple functional binding groups. Another embodiment involves a methodof detecting an analyte comprising providing a substrate including anarray of regions defining multiple cells. Each of the cells includes areaction cavity containing multiple functional binding groups anddefining a channel between a source and a drain or defining a regionbetween a pair of electrodes. In a method in accordance with thisembodiment, a voltage is applied between the source and the drain or thepair of electrodes, and a parameter indicative of an analytecharacteristic is monitored when the voltage is applied.

Another embodiment includes a process of fabricating an electricallyactive combinatorial-chemical chip for biochemical analyte detectioncomprising providing a substrate including an array of regions definingmultiple cells each including a reaction cavity. Multiple functionalbinding groups are coupled to each reaction cavity. In a process inaccordance with this embodiment, an analyte is bonded to the multiplefunctional binding groups of each reaction cavity, and an analytesensing structure is formed including the substrate with the array ofregions. Reaction cavities may be coupled with different functionalbinding groups or different molecules containing different groups.

To address the problems of creating a large number of specific probes,immobilizing them in small surface areas and applying the chips tosamples containing unknown analytes, an embodiment of the invention canadopt a “probe-less” approach. An embodiment of the invention can varysurface properties to selectively attract proteins and/or othermolecules. An embodiment of the invention can include creating a bindingsite with several small molecules (binding components). Small moleculesand/or binding components are intended to mean non-polymeric molecules(e.g., can be hetero-oligomers). To achieve this, a limited number ofbinding components (e.g., groups or molecules, covalently attached oradsorbed) can be used in different ratios and densities to obtain alarge number of different chemical matrices that have different bindingpotentials to different analytes. Biochips made by this method can betermed combinatorial chemical (CC) chips.

An embodiment of the invention can use multiple small compounds (bindingcomponents) to assemble arrays of combinatorial chemical matrices forspecific analyte binding and detections. Detections can be achievedoptically, electronically or electrically. Thus, an embodiment of theinvention can eliminate costly and time-consuming specific probegeneration and also allow detection of not-yet-identified analytes. Anembodiment of the invention is useful for sample profiling, and it isparticularly useful for the analyses of proteins as well as otherbio-analytes.

Referring to FIG. 1C, a basic element of an AECC chip embodiment of theinvention is depicted. A substrate 170 can provide structural support. Afirst binding group 181 is coupled to the substrate 170. A secondbinding group 182 is also coupled to the substrate 170 at aninter-molecular distance from the first binding group 171. An analyte190 binds to both the first binding group 181 and the second bindinggroup 182. The inter-molecular distance between the first binding group181 and the second binding group 182 corresponds to the inter-moleculardistance between the binding locations on the analyte 190. Thisembodiment of the invention is based on the principle of using differentmolecules (binding groups) for specific binding of analytes. Thisembodiment of the invention is very flexible, very compact, sensitive,fast, reasonably specific and accurate. The identification of theanalyte depends on the binding pattern of the analyte in the reactioncavities of the apparatus and prior information derived from knownanalytes

An embodiment of the invention can include: a chip surface divided intomultiple sub-areas (regions), each said sub-area can be coated with acombination of different binding components, said binding components canbe organic compounds; said different binding components can vary insize, composition, and arrangement of functional groups; the ratios anddensities of said binding components can be different among differentsub-areas and these sub-areas can be identifiable (indexed) by X-Ycoordinators.

In an embodiment of the invention, binding of an analyte on a sub-areacan require the presence of 2 or more binding components. An electricalpotential can be applied individually to or sensed individually fromeach sub-area; analyte binding can be detected electrically orelectronically; and these detection methods can be used for analyzing(profiling) of biological or chemical samples.

An embodiment of the invention can include a chip having a planarsurface with an array of sub-areas; each of the sub-areas can have 1 ormore micro or nano-wells (i.e., reaction cavities). Under each suchsub-area or well, there can be an electronic sensor and/or electricalstructures(s) (e.g., transistors or electrodes for electricaldetections). Different chemicals (for instance, 2, 3, 4 or more) can beapplied on the surface. When used in different ratios and differentdensities, a large number of combinations of chemicals (permutations)can be created. A simple way to generate different ratios is to createdifferent gradients from the binding compounds, each of the gradientscorresponding to one of the binding components.

Referring to FIG. 2, a multi-chemical-gradients (MCG) chip 200embodiment of the invention is depicted. The top portion of the figuredepicts a top plan view and the bottom portion of the figure depicts apartial cross section view. In this embodiment, A, B C and D are 4different chemical compounds. As depicted, A-B gradient(s) vary frombetween right and left and are represented by the horizontal doubleended arrow. As depicted, C-D gradient(s) vary from between top andbottom and are represented by the vertical double ended arrow. In thisembodiment, a surface 210 of a substrate 220 of the chip 200 includes anarray of regions, each of which defines a sub-area 230. Each of thesub-areas 230 includes a sensor unit 240 which in-turn includes anano-well 250 (reaction cavity) and a semiconductor or electrical sensor260.

Referring to FIGS. 3A-3C, the chemicals (e.g., compounds of the bindingcomponents and solvents/vehicles) can be delivered to the surface of thewells by printing methods. Referring to FIG. 3A, a printing head 310 iscoupled to a pair of mixers 320 each of which is in-turn coupled to apair of reservoirs 330. The printing head 310 can deliver apredetermined ratio of A/B/C/D to a substrate surface 340. Referring toFIG. 3B, a plurality of filled binding cavities 350 is arranged above aplurality of sensors 360 on the substrate. Referring to FIG. 3C,preferably, self-assembled mono-layers (SAM) 370 are formed in each well(reaction cavity 350). For example, the bottom of the well can be coatedwith gold, a thiol-polyethylene glycol (PEG) derived compound can beused as a base component and compounds with similar (or the same) basestructure(s) together with additional functional groups on the other endof the similar base structure molecules are the binding components andused together with base component to form a mixed SAM (self assembledmonolayer). The functional groups associated with the binding componentsplay the binding roles in analyte binding.

Referring to FIG. 4, four schematic examples of combinatorial chemicalstructures are depicted. The organic compounds used as bindingcomponents have different functional groups. Positively charged (PC)compounds are typically compounds with amino groups. Negatively charged(NC) compounds can be those containing carboxyl groups, sulfate groupand phosphate groups. Compounds which are hydrophobic (nonpolar (NP))can be those with benzyl ring structures and alkyl chains. Othercompounds that are hydrophilic (polar (p)) can also be used, such ascompounds with hydroxyl group, amine group, or organic compounds withhetero-atoms (e.g., nitrogen, oxygen). Organic compounds with halogenatoms can also be used. Compounds with reactive compounds groups mayalso be used, such as compounds with a thiol group, or an aldehydegroup. Short peptides, including non-natural amino acids and oligonucleotides (including those with modified structures) can be usedtogether with other organic compounds. Other factors can also beconsidered in fabricating CC chips: for example, molecular chain length,position of functional groups, distance between functional groups,number of functional groups per molecule, ratio of mixed functionalgroups per molecule, arrangement of mixed functional groups on amolecule. These factors are important in generating 3-dimensionalbinding sites.

Referring to FIG. 5, CC chip can also be made with more than 4 chemicalconditions (independent variables). For instance, the binding components(functional groups can be structurally arranged in a molecule to providea contextual condition. In this way, an additional condition can bemolecular chain length. The position of functional groups in a moleculecan be another condition. The distance between function groups can be acondition. The number of functional groups per molecule can be acondition. the ratio of mixed functional groups per molecule can be acondition. The arrangement of mixed functional groups in a molecule canalso be a condition. Also, the total density of functional groups onsurface (region) can be a condition.

Referring to FIGS. 6A-6B, 7 and 8A-8C, different electrical/electronicsensors can be used together with a CC chip. Optical sensors can also beused together with a CC chip. In the case of an activeelectrical/electronic sensor, the chip can be termed an electricallyactive CC chip or EACC chip. For example, field-effect-transistorsensors, capacitance and impedance sensors and/or static-electricsensors can be integrated in the chip. Ideally, there is a sensorassociated with each reaction cavity and each of the sensors iscontrolled independently.

Referring to FIG. 6A, a field effect measurement embodiment is depicted.An analyte 610 in a reaction cavity 620 with aqueous buffer is bonded toan SAM layer 630 to define a channel 640 on a substrate 650. The channel640 is located between a source 645 and a drain 655 which are bothcoupled to a voltage source and monitoring system 660. Referring to FIG.6B, a capacitance or impedance measurement embodiment is depicted. Theanalyte 610 is again bonded to the SAM layer 630 to define the channel640 on the substrate 650. In this embodiment, the channel 640 is locatedbetween a first electrode 670 and a second electrode 675 which are bothcoupled to a voltage source and monitoring system 660.

Referring to FIG. 7, a co-planar electrode static-electrical orcapacitance/impedance measurement embodiment is depicted. A self alignedmonolayer 710 is connected to a bottom surface electrode 720. The bottomsurface electrode 720 is coupled to a source connector 725. A topsurface electrode 730 is located opposite the self aligned monolayer 710across a reaction cavity with aqueous buffer.

Sample binding: any biochemical or chemical samples can be used,provided chips with affinity surfaces are used. Conditions for samplebinding and washing can be similar to those used in standardchromatography procedures: ion exchange, size exclusion, affinitybinding, reverse phase binding (e.g., varying pH, ionic strength,solvent concentration) Sample concentrations, binding time and washingconditions can also be modified from the standard procedures. Amicrofluidic system (or micro electromechanical system (MEMS)) can becombined with the chip.

Detection: Field effect, capacitance and impedance can be monitored foreach reaction cavity, provided suitable electrical/electronic structuresare made in the chip. An external chip reader is preferably used tocollect and analyze the data. FIGS. 8A-8C illustrate an example ofdetection based on static-electric attraction. After selective bindingand washing, an electrical potential is applied between a top surface ofthe chip 810 and a bottom surface of the chip 820. After drying byvacuum, electrical charges are built up around the molecules. Thecharges make the molecules move (fly) toward the top surface. Becausethe top plate can have a transistor (charge detector) corresponding tothose in the bottom plate, molecular charging and flying can beregulated and detected independent of those in other reaction cavities.

Several transistors can be in a cell (reaction cavity) with the gates ofthe transistors coupled to the binding molecules. An SAM layer may notbe necessary due to the importance of the distance between the analytesand the gate surface (i.e., the closer the better). The binding of theanalytes close to the gate can affect electron distribution and thus theconductance of the transistor (between source and drain). Another typeof structure in a cell (cavity) is a combination of electronic sensor(transistor) and electrical sensor (electrode for impedancemeasurement).

Data interpretation can be based on the premise that no specific probesor binding partners are required. Therefore, data obtained should becompared to reference or control samples or to normalized data.Algorithms can be trained and used to address particular problems.

Embodiments of the invention are applicable to clinical, research,pharmaceutical, agriculture, and environmental protection. Samples mayneed fractionation or enrichment before contacting a chip. Differentchips can be used for the same sample to get complete information ofinterest.

The invention can include modifying the surface of glass or silicon withsilanes that contain phenyl or other aromatic moieties that haveabsorption at about 260 nm and below. These materials can form a selfassembling monolayer (SAM) using standard microelectronics processingtechniques such as those used to promote adhesion of photoresists instandard high volume manufacturing (HVM) processing.

Referring to FIG. 9A, an embodiment of the invention is depicted asincluding a self aligned monolayer (SAM) on a substrate of silicon,silica, or metal oxides. In the case of simple aromatic groups R can be,e.g., hydrogen, amine, ethylenediamine, cyano, methyl, or fluorinegroups. Therefore, the starting SAM can have many different chemicalcharacteristics that determine the surface energy, polarity, andcapability to attach additional moieties to or just be a relativelyinert reaction well characterized starting surface for furthermodification using deep ultra violet (DUV) light. An embodiment of theinvention can use a phenyl group (R═H) for the example depicted in FIGS.9A-9D.

Once the substrate has been treated it can be exposed (flood or usinghigh resolution mask) on a standard and readily commercially availableDUV scanner or stepper. Because the Si—C bond is the weakest bond in theSAM what occurs is the breakage of that bond and the phenyl group isvolatilized. In ambient atmosphere, the Si—: radical reacts with O2 &H2O to form SiOH. It is important to note that this is the same surfaceas the initial substrate surface, but before the formation of the SAM,and it is now one Si atom taller. The dose in mJ/cm2 to completelyremove all of the phenyl groups is well documented in severalpublications and is on the order of 200 to 1000 mJ/cm2 and is alsodependent on the type of aromatic and organic group chosen for theoriginal SAM. For instance, it can be assumed that 500 mJ/cm2 is thedose to remove all the phenyl groups.

Referring to FIG. 9B, the resulting surface after the substrate and SAMhas been exposed to 50 mJ/cm2 is depicted. After exposure ˜10% of thesurface is now available for a 2nd SAM to be formed. It is mostimportant to note in this example that 2nd SAM material may have noaromatic group and, therefore, will not be affected by subsequent DUVexposures because it has substantially no absorption in the DUVspectrum, i.e., above ˜200 nm. In this example,perflourooctyldimethylchlorosilane is used (SAM2) as the next SAMformation material. Treatment of the exposed surface with SAM2 willyield a new surface containing ˜10% of SAM2 and 90% of the originalphenyl silane SAM as depicted in FIG. 9C.

In this example, one additional exposure/SAM formation using atrimethoxysilane N-(2-aminoethyl-3-aminopropyl) trimethoxysilane SAM3 isperformed, but this process could be continued to build a very largevariety of well defined surfaces. In this example a 2nd exposure is 100mJ and will remove ˜20% of the remaining phenyl groups and followingtreatment with SAM3 will create a surface with ˜20% SAM3 ˜10% SAM2 and˜70% of the original SAM as depicted in FIG. 9D.

This procedure could be continued to put more SAMs of knownconcentration on the surface and subsequent surface chemistry can bedone to attach bio-relevant chemistry such as antibodies, DNA, or RNA,to the appropriate R group on the SAM. The surface can thus be patternedin arrays very easily and even have high resolution (<100 nm line/space)within an array. This embodiment of the invention makes it feasible tomake arrays of well defined surface chemistry with minimal reticles ormasks.

For instance, if it were desired to make small areas (100 um square) ofwell defined, but different surface concentrations of the three SAMsdescribed above on bare Si metal oxides or glass, the followingtechnique could be used. The surface can be treated with photosensitivetrichlorophenylsilane and then exposed via a 10 um×10 um array with doseincrements of 5 mJ/cm2 (i.e., ˜1% the does assumed above to be requiredto remove all the phenyl groups) over a range from 0-500 mJ/cm2. ThenSAM2 formation can be performed resulting in 10×10 array containing aratio of the 1^(st) two SAMs of from approximately 0% to approximately100% across the array. The 2^(nd) exposure can then be performed but inreverse spatial arrangement of the increments, or with any desired doserange, to yield many different surfaces of known composition.Specifically, with a reverse exposure starting at 500 mJ and going to 0in the same increments, the result would be 10×10 array that contains˜100% SAM2 & SAM3, but with ˜0% of the original SAM.

However, in another instance, an embodiment of the invention couldutilize the same range and not reverse dose, and this would result in anarray with ˜100% original SAM and with the SAM2 & SAM3 increasing inconcentration by 1% each until they reach ˜50% each of the surfaceconcentration half way through the array. From then on SAM2 wouldcontinue to increase by 1% and SAM3 would decrease by 1% with ˜0% oforiginal same making up the concentration of the surface until 100% SAM2is reached at last exposure field. The variations on this sub-genericscenario are enormous. The same examples described above can work with193 nm exposure which will make the aromatic photosensitive SAMs moreefficient but will also make many of the non-aromatic SAMs slightlysensitive to each of the subsequent exposures, but since they are somuch less absorbing they will be much less involved in the photochemicalcleavage, and therefore they are just accounted for in determining thefinal composition of the surface.

While not being limited to any particular performance indicator ordiagnostic identifier, preferred embodiments of the sensor array can beidentified one at a time by testing for the presence of sensing withrespect to a known concentration of target analyte. The test for thepresence of sensing can be carried out without undue experimentation bythe use of a simple and conventional impedance spectroscopy experiment.Among the other ways in which to seek embodiments having the attributeof sensing guidance toward the next preferred embodiment can be based onthe presence of a characteristic IR spectroscopy signal.

Embodiments of the electrically active combinatorial-chemical chip forbiochemical analyte detection can be identified by scanning electronmicroscope (SEM) cross-sections. Embodiments of the electrically activecombinatorial-chemical chip for biochemical analyte detection can alsobe identified by material analysis of devices containing sensors usingtechniques such as Auger spectroscopy and/or dynamic secondary ion massspectroscopy.

Embodiments of the invention can include impedance spectroscopy,amperommetry, voltammetry and other electrochemical techniques used togenerate a response from adsorbed analyte through the electrodes/probes.Embodiments of the invention can include the use of optical techniquessuch as FTIR spectroscopy can be used to identify the functional groupsof analyzed chemicals species.

Specific embodiments of the invention will now be further described bythe following, nonlimiting examples which will serve to illustrate insome detail various features. The following examples are included tofacilitate an understanding of ways in which embodiments of theinvention may be practiced. It should be appreciated that the exampleswhich follow represent embodiments discovered to function well in thepractice of embodiments of the invention, and thus can be considered toconstitute preferred modes for the practice of embodiments of theinvention. However, it should be appreciated that many changes can bemade in the exemplary embodiments which are disclosed while stillobtaining like or similar result without departing from the spirit andscope of embodiments of the invention. Accordingly, the examples shouldnot be construed as limiting the scope of embodiments of the invention.

EXAMPLES Example 1

Referring to FIGS. 10A and 10B, a DNA-based self-assembly structure isprovided for affinity binding array. A single-stranded oligonucleotidewith one or more “coding regions” and a binding ligand A1 is immobilizedon one of the spots on an array surface (FIG. 10A). The binding ligandspreferably include small molecules such as biotin, pyridine, furan,imidazole, pyran, benzene, purine, pyrimidine, benzoic acid, aniline,styrene, phenol, typtophan, or another compound of interest or as may beunderstood by those skilled in the art. The oligo nucleotide is fromapproximately 20 to approximately 100 bases long, and each coding regioncontains from approximately 10 to approximately 20 DNA bases withspecific sequences; while the binding ligands are small molecules suchas biotin, pyridine, furan, imidazole, pyran, benzene, purine,pyrimidine, benzoic acid, aniline, styrene, phenol, typtophan, or anyother compounds of interest. The ligand can be attached to theoligonucleotide through known chemistry, e.g., N-hydroxysuccinimideester (NHS) mediated conjugation (FIG. 10B),1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide (EDC) catalyzed amideformation or reductive amination.

In general, there are several ways to immobilize DNA. A first would becharge attraction on a positively charged surface such as a surfacecoated with polysine. A second would be covalent attachment through amolecular end such as a thiol attachment reaction with a metal. An aminegroup on DNA will react with a carboxyl group on a surface. A third wayinvolves specific binding. For example, biotin on DNA may be captured bystreptavidin on the surface.

After the immobilization, a second single-stranded oligonucleotide withone or more “coding regions” and ligand B1 was contacted with thesubstrate. The oligo nucleotide can be 20-100 bases long, and eachcoding regions can contains 10-20 DNA bases with specific sequences Oneof the coding regions of the second oligonucleotide should becomplimentary to one of the coding region. Under hybridization condition(for example, incubation at 37° C. for 1 hour in 2×SSC buffer: 0.03 Msodium citrate, 0.3 M NaCl, pH approx. 7.0,) the two oligo nucleotideswill hybridize and thus the two ligands A1 and B1 will be localized.

Additional steps can be performed to add more ligand-oligonucleotides tothe surface, resulted in a collection of ligands A1, B1, C1 . . .localized in the said array spot at certain orientation. On other arrayspots, different ligands or different combination of ligands can beapplied to create another unique collection of ligands. Thus, anaffinity array based on DNA self-assembly can be generated.

Solvents that can be used in the ligand incorporation step and thespotting step include water, salt buffer solution such as SSC, andorganic solvent in which oligonucleotide is soluble, such as methanoland DMF (dimethyl Formamide). For the hybridization step, buffersolutions such as SSC, citrate, borate and phosphate with up to 50% ofFormamide or urea can be used. The same format can be used to make PNAand RNA self-assembly arrays as well.

Example 2

Referring to FIG. 11, an embodiment of the invention includes across-linked polymer based structure for affinity binding array. Smallmolecule ligand A1 was incorporated into a cross-linkable polymers suchas poly(acrylamide)-co-poly(acrylic acid), and other ligand B1, C1, wasincorporated into other cross-linkable polymers individually. Thecross-linkable polymer was selected from synthetic polymers such aspolyacrylamide, polyacrylic acid, polyallyamine, polyvinylalcohol, andnatural polymers such as polysaccharide and DNA.

Referring to FIGS. 12A and 12B, two possible incorporation methods areco-polymerization (FIG. 12A) and chain-transfer (FIG. 12B). In theco-polymerization method, a reactive monomer was added to thepolymerization mix and the resulted co-polymer was reacted with ligandA1, B1, through the reactive function group NHS-ester. In the chaintransfer method, the ligands were linked to a chain-transfer reagentsuch as thiol, and hence incorporated at the end of the polymer chain.Other polymer functionalization methods such as post-polymerizationreaction can also be used.

A mixture of the ligand-loaded cross-linkable polymer was deposited onan array spot, and a cross-linker such as 1,4-diaminobutane wasintroduced after the polymers were activated by EDC(1-Ethyl-3-(3-dimethyllaminopropyl)carbodiimide). The resultingcross-linked polymer had the attached ligands (A1, B1, C1 . . . )localized in the array spot. Using this method, each array spot wasdeposited with a different set of ligands, and an affinity array wasbuilt.

Preferred and alternative solvents that were and/or could be used in theligand incorporation step, the spotting step, and cross-linking stepsinclude water-based solvents such as water and buffer solutions such ascitrate, borate and phosphate; and organic solvent such as DMF (dimethylFormamide), ethyl alcohol, methanol, iso-propanol, and THF(tetrahydrofuran). Other cross-linking methods such as glutaraldehydecrosslinking of amines or photo-initiated radical crosslinking ofpoly-acrylamide can also be used.

Example 3

Referring to FIGS. 13A-13D, an embodiment of the invention includesmaking combinatory chemical structures. PEG-based thiol compounds withcombinatory chemical structures were made from thiol-PEG-amine orthiol-PEG-carboxylate. DCC=N,N′-dicyclohexylcarbodiimide. The molecularweight of these thiol-PEG chemical structures was from 200 to 5000, with3-100 repetitive PEG units. The above reactions were performed in water,water-based buffer solution such as sodium phosphate solution, andorganic solvents such as DMF and THF.

Generally, PEG polymers may be advantageously used, as in example 3,primarily to fill the surface spaces or gaps on a chip. It can reducenon-specific binding. If one end of the PEG polymers have thiol groupsthat can be attached to a gold surface, the PEG molecules canself-assemble into a monolayer on the gold surface. If a portion of thePEG polymers have functional groups (for example, examine groups), thenthe chip surface will be functionalized by the amine groups whosedensity is determined as a % of the amine-containing PEG polymers.

A practical application of embodiments of the invention that has valuewithin the technological arts is integrating chemical and/or biologicalsensing with computing and communication. There are virtuallyinnumerable uses for embodiments of this invention.

Embodiments of the invention are cost effective and advantageous for atleast the following reasons. An embodiment of the invention obviates theneed for specific probe synthesis as well as site-specificimmobilization and only a limited number of small chemicals can be usedin different combinations. An embodiment of the invention can have theadvantage of simplifying chip fabrication. An embodiment of theinvention can have a compact size and high capacity. Electricaldetection allows fabrication of chips with very high sub-area density(protein chip with feature size <1 um), no optical system and thus chipreader can be very compact. An embodiment of the invention can beflexible and applicable to samples of different compositions and fromdifferent sources. An embodiment of the invention can have the advantageof detecting not-yet-identified analytes in that unknown compounds canbind to the EACC chips and be detected. In general, embodiments of theinvention improve quality and/or reduce costs compared to previousapproaches.

The terms “a” or “an”, as used herein, are defined as including one andmore than one. The term plurality, as used herein, is defined asincluding two and more than two. The term another, as used herein, isdefined as at least a second or more. The terms “comprising” (comprises,comprised), “including” (includes, included) and/or “having” (has, had),as used herein, are defined as open language (e.g., requiring what isthereafter recited, but open for the inclusion of unspecifiedprocedure(s), structure(s) and/or ingredient(s) even in major amounts.The terms “consisting” (consists, consisted) and/or “composing”(composes, composed), as used herein, close the recited method,apparatus or composition to the inclusion of procedures, structure(s)and/or ingredient(s) other than those recited except for ancillaries,adjuncts and/or impurities ordinarily associated therewith. The recitalof the term “essentially” along with the terms “consisting” or“composing” renders the recited method, apparatus and/or compositionopen only for the inclusion of unspecified procedure(s), structure(s)and/or ingredient(s) which do not materially affect the basic novelcharacteristics of the composition. The term coupled, as used herein, isdefined as connected, although not necessarily directly, and notnecessarily mechanically. The term any, as used herein, is defined asall applicable members of a set or at least a subset of all applicablemembers of the set. The term approximately, as used herein, is definedas at least close to a given value (e.g., preferably within 10% of, morepreferably within 1% of, and most preferably within 0.1% of). The termsubstantially, as used herein, is defined as largely but not necessarilywholly that which is specified. The term generally, as used herein, isdefined as at least approaching a given state. The term deploying, asused herein, is defined as designing, building, shipping, installingand/or operating. The term means, as used herein, is defined ashardware, firmware and/or software for achieving a result. The termprogram or phrase computer program, as used herein, is defined as asequence of instructions designed for execution on a computer system. Aprogram, or computer program, may include a subroutine, a function, aprocedure, an object method, an object implementation, an executableapplication, an applet, a servlet, a source code, an object code, ashared library/dynamic load library and/or other sequence ofinstructions designed for execution on a computer or computer system.

All the disclosed embodiments of the invention disclosed herein can bemade and used without undue experimentation in light of the disclosure.Embodiments of the invention are not limited by theoretical statementsrecited herein. Although the best mode of carrying out embodiments ofthe invention contemplated by the inventor(s) is disclosed, practice ofthe embodiments of the invention is not limited thereto. Accordingly, itwill be appreciated by those skilled in the art that the embodiments ofthe invention may be practiced otherwise than as specifically describedherein.

It will be manifest that various substitutions, modifications, additionsand/or rearrangements of the features of the embodiments of theinvention may be made without deviating from the spirit and/or scope ofthe underlying inventive concept. It is deemed that the spirit and/orscope of the underlying inventive concept as defined by the appendedclaims and their equivalents cover all such substitutions,modifications, additions and/or rearrangements.

All the disclosed elements and features of each disclosed embodiment canbe combined with, or substituted for, the disclosed elements andfeatures of every other disclosed embodiment except where such elementsor features are mutually exclusive. Variation may be made in the stepsor in the sequence of steps defining methods described herein.

Although the sensor array described herein can be a separate module, itwill be manifest that the sensor array(s) may be integrated into thesystem with which it is (they are) associated. Similarly, although thehand held device described herein can be a separate module, it will bemanifest that the hand held device(s) may be integrated into the systemwith which it is (they are) associated.

The sensor array may comprise an array of transistor sensors. Differentchemical structures (groups or molecules) may be disposed on the gatesof the transistors. A sample preferably contacts each of thegate-associated chemical structures. A set of pre-determined chemicalstructures are associated with a set of transistors. Different analytesinteract with the pre-determined chemical structures differently, suchthat the patterns are unique. Binding patterns are translated intoelectrical signals by the transistors. The analytes in the sample may beidentified by the pattern of electrical signals of the transistors withrespect to the gate-associated chemical compositions. A database ispreferably pre-built using standard analytes, and computer patternrecognition is used in the identification.

The individual components need not be formed in the disclosed shapes, orcombined in the disclosed configurations, but could be provided in allshapes, and/or combined in all configurations. The individual componentsneed not be fabricated from the disclosed materials, but could befabricated from all suitable materials. Homologous replacements may besubstituted for the substances described herein. Agents that are bothchemically and physiologically related may be substituted for the agentsdescribed herein where the same or similar results would be achieved.

While an exemplary drawings and specific embodiments of the presentinvention have been described and illustrated, it is to be understoodthat that the scope of the present invention is not to be limited to theparticular embodiments discussed. Thus, the embodiments shall beregarded as illustrative rather than restrictive, and it should beunderstood that variations may be made in those embodiments by workersskilled in the arts without departing from the scope of the presentinvention as set forth in the appended claims and structural andfunctional equivalents thereof.

In methods that may be performed according to the invention and/orpreferred embodiments herein and that may have been described aboveand/or claimed below, the operations have been described in selectedtypographical sequences. However, the sequences have been selected andso ordered for typographical convenience and are not intended to implyany particular order for performing the operations.

The appended claims are not to be interpreted as includingmeans-plus-function limitations, unless such a limitation is explicitlyrecited in a given claim using the phrase(s) “means for” and/or “stepfor.” Subgeneric embodiments of the invention are delineated by theappended independent claims and their equivalents. Specific embodimentsof the invention are differentiated by the appended dependent claims andtheir equivalents.

1. A method of detecting a biochemical analyte comprising: providing asubstrate including an array of regions defining a plurality of cells,each of the plurality of cells including a reaction cavity comprising asensor comprising at least one transistor with a source, a drain, and agate; and multiple functional binding groups on the gate of saidtransistor, wherein the array of regions comprises a first gradient of afirst functional binding group in a first direction of the array ofregions and a second gradient of a second functional binding group in asecond direction of the array of regions and wherein the multiplefunctional binding groups in the plurality of cells are in differentratios and/or densities; wherein a distance between the first bindinggroup and the second binding group corresponds to an inter-moleculardistance between binding locations on the biochemical analyte;contacting a sample containing said biochemical analyte onto saidsubstrate, thereby allowing said biochemical analyte to interact withthe multiple functional binding groups; applying a voltage between thetransistor's source and drain; detecting an electrical parameter in atleast one reaction cavity to thereby detect said biochemical analyte. 2.The method of claim 1, further comprising identifying the analyte by asignal pattern of the electrical parameter from the plurality of cellsand wherein the signal pattern is compared to reference signal patternsthat were generated from known analytes.
 3. The method of claim 2,wherein the reference electrical parameter signal patterns of knownanalytes were previously-obtained or retrieved from a database.
 4. Themethod of claim 1, wherein each of the plurality of cells comprises ananalyte bonded to a self-assembled monolayer.
 5. The method of claim 4,wherein said analyte bonded to said self-assembled monolayer defines achannel.
 6. The method of claim 1, wherein the substrate surface ismodified with silane.
 7. The method of claim 1, wherein each sensor isindependently addressable.
 8. The method of claim 1, further comprisingapplying an electrical potential between the source and the drain duringthe detecting the electrical parameter.
 9. The method of claim 1,wherein the electrical parameter is a change in capacitance or impedanceafter the analyte binds to a gate.
 10. The method of claim 1, whereinthe first and second directions are orthogonal.
 11. The method of claim1, wherein the array of regions comprises a third gradient of a thirdfunctional binding group in a third direction of the array of regionsand a fourth gradient of a fourth functional binding group in a fourthdirection of the array of regions.
 12. The method of claim 1, whereinthe third and fourth directions are opposite to the first and seconddirections.
 13. A method of detecting a biochemical analyte comprising:providing a substrate including an array of regions defining a pluralityof cells, each of the plurality of cells including a reaction cavitycomprising a sensor comprising at least one transistor with a source, adrain, and a gate, the reaction cavity containing multiple functionalbinding groups between a pair of electrodes, wherein the array ofregions comprises a first gradient of a first functional binding groupin a first direction of the array of regions and a second gradient of asecond functional binding group in a second direction of the array ofregions and wherein the multiple functional binding groups in theplurality of cells are in different ratios and/or densities; wherein adistance between the first binding group and the second binding groupcorresponds to an inter-molecular distance between binding locations onthe biochemical analyte; contacting a sample containing said biochemicalanalyte onto said substrate, thereby allowing said biochemical analyteto interact with the multiple functional binding groups; applying avoltage between the pair of electrodes; detecting an electricalparameter in at least one reaction cavity to thereby detect saidbiochemical analyte.
 14. The method of claim 13, further comprisingidentifying the analyte by a signal pattern of the electrical parameterfrom the plurality of cells and wherein the signal pattern is comparedto reference signal patterns that were generated from known analytes.15. The method of claim 14, wherein the reference electrical parametersignal patterns of known analytes were previously-obtained or retrievedfrom a database.
 16. The method of claim 13, wherein each of theplurality of cells comprises an analyte bonded to a self-assembledmonolayer.
 17. The method of claim 16, wherein said analyte bonded tosaid self-assembled monolayer defines a reaction cavity region betweenthe pair of electrodes.
 18. The method of claim 13, wherein thesubstrate surface is modified with a silane.
 19. The method of claim 13,wherein each sensor is independently addressable.
 20. The method ofclaim 13, further comprising applying an electrical potential betweenthe source and the drain during the detecting the electrical parameter.21. The method of claim 1, wherein the electrical parameter is a changein capacitance or impedance after the analyte binds to a gate.
 22. Themethod of claim 13, wherein the first and second directions areorthogonal.
 23. The method of claim 13, wherein the array of regionscomprises a third gradient of a third functional binding group in athird direction of the array of regions and a fourth gradient of afourth functional binding group in a fourth direction of the array ofregions.
 24. The method of claim 13, wherein the third and fourthdirections are opposite to the first and second directions.